Throughout this application various publications are referenced by numbers within parentheses. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The human ras gene family consists of three members: the H-ras, K-ras and the N-ras gene (1). These genes code for related proteins of 21 kD, which are located at the inner face of the cell membrane (36) and are thought to be involved in transducing signals from cell surface receptors to their intracellular targets (37). A significant portion of tumor cell lines and fresh tumor tissue has been found to possess an activated ras gene. Such genes are characterized by their ability to induce oncogenic transformation of mouse 3T3 cells. In most cases so far analyzed the activation is due to a point mutation in the 12th or 61st codon of a ras gene resulting in a single amino acid substitution in the gene product (3-15).
The fixed position of the mutations makes it possible to use synthetic oligonucleotides to screen directly for the presence of activating mutations in genomic DNA of tumor tissue. This direct assay is based on the fact that a fully matched DNA hybrid has a higher thermal stability than a hybrid with a mismatched basepair. By using a selective washing procedure nonperfect hybrids can melt off, whereas a fully matched hybrid remains stable.
This type of assay has been introduced by Conner et al. (24) to detect a single base mutation in the betaglobin gene. In a previous study, differential hybridization with synthetic oligonucleotides has been used to detect point mutations induced by chemical carcinogens in codon 12 of Ha-ras-1 in rat DNA (44). Similarly, this approach has been used to demonstrate a single base change in the mouse K-ras oncogene in codon 12 in tumors induced by gamma radiation (45). To detect possible mutations that lead to amino acid substitution at position 12 or 61 of the three ras genes, H ras, K-ras and N-ras, we have synthesized sets of 20-mers which can detect whether and at which position a mutation is present. One embodiment of this invention concerns an assay system for mutations in codon 12 and 61 of the human ras gene.
The use of focus assays to detect morphological transformation of NIH 3T3 cells in DNA transfection experiments has shown that between 10-30% of human tumors contain altered forms of either the c-H-ras-1 gene, the c-K-ras-2 gene or the N-ras gene. These genes encode GTP binding proteins of 188 and 189 amino acids which are located at the inner surface of the cell membrane and are highly conserved in evolution (1,2). In 15 or so cases of altered ras genes in human tumors or in human tumor cell lines that have been analyzed so far, activation has been shown to be the result of a single basepair mutation which leads to the substitution of either glycine at amino acid position 12 or glutamine at position 61 (3-17). However, recent in vitro mutagenesis experiments indicated that mutations at amino acids 13, 59, and 63 can also lead to transforming activity (18).
We have studied the presence of activated ras genes in Acute myeloid leukemia (AML). This leukemia is characterized by abnormal proliferation and differentiation of cells of the myeloid, monocytic and erythroid lineage. It is subdivided in 6 groups, FAB M1-M6, (19) depending on which immature cell is dominant in population. Using a direct in vivo selection assay in nude mice of transfected 3T3 cells (20) we have found that 4 out of 5 AML DNAs tested contain an activated N-ras gene. An altered N-ras oncogene was also detected in another AML sample using the focus formation assay. These activated N-ras genes were analyzed for the presence of mutations using a set of synthetic oligonucleotide probes and it was found that all 5 activated N-ras genes contain a mutation at codon 13. One embodiment of this invention concerns the use of these oligonucleotide probes to detect a mutation at codon 13 of the N-ras gene. Detection of such a mutation is useful in the diagnosis of certain types of AML and other tumors.